Combining Metabolic Engineering and Lipid Droplet Assembly to Achieve Campesterol Overproduction in Saccharomyces cerevisiae

Abstract

Campesterol is a kind of important functional food additive. Therefore, stable and efficient campesterol biosynthesis is significant. Herein, we first knocked out the sterol 22-desaturase gene in Saccharomyces cerevisiae and expressed sterol Δ7-reductase from Pangasianodon hypophthalmus, obtaining a strain that produced 6.6 mg/L campesterol. Then, the modular expression of campesterol synthesis enzymes was performed, and a campesterol titer of 88.3 mg/L was achieved. Because campesterol is a lipidsoluble macromolecule, we promoted lipid droplet formation by exploring regulatory factors, and campesterol production was improved to 169.20 mg/L. Next, triacylglycerol lipase was used to achieve compartment campesterol synthesis. After enhancing the expression of sterol Δ7-reductase and screening cations, the campesterol titer reached 438.28 mg/L in a shake flask and 1.44 g/L in a 5 L bioreactor, which represents the highest campesterol titer reported to date. Metabolic regulation combined with lipid droplet engineering may be useful for the synthesis of other steroids as well.

Efficient production of 22(R)‐hydroxycholesterol via combination optimization of Saccharomyces cerevisiae

“…[3,18] Saccharomyces cerevisiae, an attractive biochemical production host, has been used to efficiently produce terpenes and steroids through metabolic engineering strategies. [16,[19][20][21][22][23][24][25] It is of note that S. cerevisiae possesses the biosynthesis pathway for ergosterol production, which can provide precursors for the 22(R)-HCHO production (Figure 1). Three heterologous enzymes involved in 22(R)-HCHO biosynthesis in S. cerevisiae have been reported, including Dhcr24, Dhcr7, and cholesterol 22-hydroxylase.…”

“…[26] Several researchers tested Dhcr7s from different organisms to identify their ability to produce campesterol, 24-methylene-cholesterol, and diosgenin. [21,22,27] In addition, previous studies have identified cholesterol 22-hydroxylases from animals and plants by tran-scriptome analysis and homologous sequence alignment, including HsCyp11a1 (Homo sapiens), DrCyp11a1 (Danio rerio), MmCyp11a1 (Mus musculus), DzCyp90b71 (Dioscorea zingiberensis), PpCyp90b27 (Paris polyphylla), StPga2 (Solanum tuberosum), and VcCyp90b27 (Veratrum californicum). [3,7,[28][29][30][31][32] These studies have established the foundation for the heterologous production of 22(R)-HCHO.…”

“…Three heterologous enzymes involved in 22(R)-HCHO biosynthesis in S. cerevisiae have been reported, including Dhcr24, Dhcr7, and cholesterol 22-hydroxylase. [21,22,26,27] For instance, Guo et al tested 11 Dhcr24s, and found that the strain expressing DHCR24 from Gallus gallus achieved the highest 7-dehydrocholesterol accumulation. [26] Several researchers tested Dhcr7s from different organisms to identify their ability to produce campesterol, 24-methylene-cholesterol, and diosgenin.…”